Gastrointestinal effects of Nigella sativa and its main constituent, thymoquinone: a review.

Gastrointestinal (GI) diseases affect a large number of people all over the world. Uncontrolled acid secretion and occurrence of gastric ulcers are common disorders of GI tract which pose serious problems to human health. Many synthetic drugs have been used to treat GI disorders but a definite cure has not been discovered so far and the available medications cause several side effects. Nigella sativa (N. sativa) (Ranunculacea) has several therapeutic effects which are attributed to its constituents like nigellicine, nigellidine, thymoquinone, dithymoquinone, thymol and carvacrol. Several beneﬁcial pharmacological properties of this plant such as anti-oxidant, anti-bacterial, anti-histaminic, anti-hypertensive, hypoglycemic, anti-fungal, anti-inﬂammatory, anti-cancer and immunomodulatory effects were reported and different therapeutic properties such as reliving bronchial asthma, jaundice, hydrophobia, paralysis, conjunctivitis, piles, skin diseases, anorexia, headache, dysentery, infections, obesity, back pain, hypertension and gastrointestinal problems, have been described for the seeds of N. sativa and its oil. The present review provides a detailed summery of scientific researches regarding gastrointestinal effect of N. sativa and its main constituent, thymoquinone.


Introduction
Gastrointestinal diseases refer to conditions involving the esophagus, stomach, small intestine, large intestine, rectum, liver, gallbladder and pancreas. Symptoms of gastrointestinal disorders include pain, heartburn, dyspepsia, abdominal distension, nausea, vomiting, bloating, constipation and diarrhea. Among these, functional and motility disorders are the most common conditions seen in clinical practice and among the general population. The prevalence of these disorders in western countries is about 10-20%. Gastrointestinal diseases are chronic conditions which need long term medication and decrease the quality of life (Drossman, 1993). Commonly used drugs have several side effects like osteoporosis, disturbance in small intestine flora, kidney stones, anemia and increased chance of occurrence of drug-induced diseases such as gastric cancer. Therefore, due to the side effects of conventional medicine, the use of natural products in the treatment of various diseases has been in the center of attention in the last few decades.
Herbal medicine has been traditionally used for the treatment of different ailments. Nigella sativa (N. sativa) is used as an important drug in the traditional medicine like Unani and Ayurveda (Goreja, 2003;Sharma et al., 2005). Almost 80% of the populations of developing countries rely mainly on herbal medicine in primary medical therapy (Mantle et al., 2000). Medicinal plants are the source of enormous drugs and many important drugs were derived directly or indirectly from plants or from molecules of plant origin.
N. sativa, commonly known as black seed or black cumin, is an annual flowering plant from the family of Ranunculaceae, which is native to southern Europe, North Africa and Southwest Asia. N. sativa seeds include oil, protein, carbohydrate, fiber, saponin, moisture and the oil extracted from N. sativa is mostly consisted of linoleic acid, oleic, dihomolinoleic acid, palmitic acid stearic acid, myristic acid, stroles and eicodadienoic acid (El-Tahir and Bakeet, 2006).
In last three decades, numerous researches have been done to identify plantderived natural substances and understand the mechanisms of their pharmacological actions. N. sativa extract increases the activity of antioxidant enzymes (catalase, glutathione peroxidase, and glutathione-stransferase) and acts as a free radical scavenger. As an anti-cancer agent, its effects such as modulation of the activities of molecular targets including p53, p73, PTEN, STAT3, PPAR-g, activation of caspases, and generation of ROS have been demonstrated.
It also suppresses inflammatory mediators, leukotrienes, prostaglandins, and B cell-mediated immune response while balances Th1/ Th2 responses and potentiates T cell and natural killer cell-mediated immune responses (Gholamnezhad et al., 2014), as an antiinflammatory and immunomodulatory agent. In this regard, several studies demonstrated that N. sativa has anti-cancer, hepatoprotective, anti-bacterial, antischistosomiasis, anti-inflammatory and antioxidant activities in gastrointestinal system (Gholamnezhad et al., 2015).
In this review, the gastrointestinal effects of N. sativa and thymoquinone (TQ) were reviewed.

Method
To collect the related data on gastrointestinal effects of N. sativa and its main constituent, TQ, online literature resources including Medline, Pubmed, Science Direct, Scopus, and Google Scholar websites was checked from 1989 to 2015.
Colorectal cancers (development of cancer in the colon or rectum) start as a polypa growth that starts in the inner lining of the colon or rectum and progresses toward the center. The preventive effect of N. sativa oil on rat colon cancer induced by 1,2dimethylhydrazine was investigated. The animals were divided into four groups: Group 1 served as control; Group 2 received oil at post-initiation stage; Group 3 received oil at the initiation stage and Group 4 received 0.9% saline and oil from the beginning until the end of the study. The results of this study showed that N. sativa oil significantly reduced the total number of aberrant crypt foci in the postinitiation stage (group 2) whereas it showed no significant inhibitory effect on initiation stage (group 3). The results indicated that N. sativa oil has potent preventive effect on colon carcinogenesis in the post-initiation stage (Salim and Fukushima, 2003).
The preventive effect of TQ, the main constituent of N. sativa on HCT-116 human colorectal cancer cells was evaluated. The results showed that TQ is a potent agent against colon cancer cells and triggers apoptosis via a p53-dependent mechanism (Gali-Muhtasib et al., 2004). On the other hand, another study revealed that TQ has no effect against HEp-2 cancer cells (Rooney and Ryan, 2005).
The effect of TQ on pancreatic cancer cells and mucin 4 (MUC4) expressions was evaluated. MUC4 is expressed in pancreatic cancer and it contributes to the regulation of differentiation, proliferation, metastasis, invasiveness, migration, and motility of malignant cells (Chaturvedi et al., 2007;Singh et al., 2004). The results showed that TQ has cytotoxic effects against pancreatic cancer cell line FG/COLO357 and downregulated MUC4 expression through JNK and p38 MAPK pathways in a dose (0-100 µmol/L) and time-dependent manner (Torres et al., 2010).
Also, another study reported that pretreatment of pancreatic cancer cells with TQ (25 Mmol/L) for 48 h followed by gemcitabine or oxaliplatin, reduced growth of cancer cells (Banerjee et al., 2009).
The effect of TQ (4 mg/kg/day) on diethylnitrosamine -induced hepatic carcinogenesis in rats was also studied. Findings documented that TQ could inhibit the development of DENA-induced liver cancer via decreasing oxidative stress and preserving the activity and expression of antioxidant enzymes .
The anti-cancer effects of N. sativa and TQ were summarized in Table 1.

Hepatoprotective effect
The protective effect of N. sativa (0.2 mL/kg, intraperitoneal: i.p.) against hepatic ischemia/reperfusion injury was investigated in rats. Levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), total antioxidant capacity (TAC), catalase (CAT), total oxidative status (TOS), oxidative stress index (OSI) and myeloperoxidase (MPO) were measured. The results showed that N. sativa has a potential effect against hepatic ischemia/reperfusion injury and could act as a potent antioxidant agent (Yildiz et al., 2008).
In another study, the effects of N. sativa (0.2 mL/ kg, i.p.) on cholestatic liver injury were evaluated in rats. The authors found that N. sativa has a preventing effect on cholestatic liver injury in rats. The results also suggested that the reduction of neutrophil infiltration and oxidative stress in the liver was probably responsible for this protective effect (Coban et al., 2010).
In addition, the protective effect of N. sativa seeds (5% of the diet weight) against lead acetate-induced liver toxicity was documented in male rats. N. sativa seeds caused significant elevation in AST, improved biochemical and histopathological profiles and reduced damage areas (Farrag et al., 2007).
The protective effects of N. sativa oil (0. 2 mL/kg, i.p.) and Urticadioica oil (2 mL/kg, i.p.) on carbon tetrachloride (CCl4)-induced liver toxicity were studied in rats. Findings showed that N. sativa and U. dioica reduced lipid peroxidation and liver enzymes, and enhanced antioxidant defense system activity in CCl4-treated rats (Kanter et al., 2005a).
The effect of TQ (10 mg/kg, orally) on hepato-renal dysfunction, CYP3A1 and spermidine/spermine N-1-acetyl-transferase gene expression induced by renal ischaemia/reperfusion was evaluated in rats. According to this study, TQ has a protective action on renal ischaemia/reperfusion-induced damage via an antioxidant mechanism and could decrease CYP3A1 and SSAT gene expression (Awad et al., 2011).
The protective effect of TQ against tertbutyl hydroperoxide toxicity was evaluated in isolated rat hepatocytes. The results showed that pre-treatment of hepatocytes with 1 mM TQ reduced the leakage of cytosolic enzymes, ALT and AST (Daba and Abdel-Rahman, 1998).
Oral administration of a single dose (100 mg/Kg) of TQ to male Swiss albino mice resulted in a protective effect against CCl4-induced hepatotoxicity which was probably due to the antioxidant property of TQ (Nagi et al., 1999).
In another study, the protective effect of TQ (4.5, 9 and 18 mg/kg, i.p.) on Aflatoxin B1 -induced liver toxicity was evaluated in mice. Findings of this study showed that TQ significantly decreased AST, ALT, ALP and MDA levels. This protective effect may be mediated through increased resistance to oxidative stress as well as reduction in lipid peroxidation (Nili-Ahmadabadi et al., 2011).
TQ (10 mg/kg, oral) protective effect on sodium fluoride-induced hepatotoxicity and oxidative stress in rats was shown as it improved the antioxidant status and reduced the alterations in biochemical parameters. This protective effect was perhaps due to the ability of TQ to antagonize increased lipid peroxidation (LPO) and in turn stabilizing the integrity of the cellular membranes and decreasing the leakage of liver enzymes (Abdel-Wahab, 2013). Also, it was shown that TQ (50 mg/kg body weight) significantly inhibited tamoxifen-induced hepatic glutathione depletion and normalized the activity of SOD (Suddek, 2014). TQ (0.5, 1 and 2mg/kg/day, oral) combated against acetaminophen-induced hepatotoxicity and decreased acetaminophen-induced hepatotoxicity in a dose-dependent manner as evidenced by reduction in serum ALT activities. Hepatoprotective effect of TQ was probably mediated by increased resistance to oxidative and nitrosative stress and improved mitochondrial energy production . In a clinical study, the effects of ethanolic extracts of N. sativa, Zingiber officinale (Z. officinale) and their mixture were evaluated in patients with hepatitis C virus (HCV) infection. Patients were divided into five groups: I) Healthy subjects, II) HCV control; III) HCV patients receiving a capsule containing 500 mg N. sativa extract twice daily; IV) HCV patients receiving a capsule containing 500 mg Z. officinale extract twice daily and V) HCV patients receiving a capsule containing 500 mg Z. officinale and 500 mg N. sativa extracts twice daily. The results showed that ethanolic extracts of N. sativa and Z. officinale had a significant effect in HCV patients as shown by a decrease in viral load and restoration of liver functions (Adel et al., 2013).
The hepatoprotective effects of N. sativa and TQ were summarized in Table 2.

Hepatorenal dysfunction induced by renal ischaemiareperfusion
Reduced damage via an antioxidant mechanism Reduced of CYP3A1 and SSAT gene expression (Awad et al., 2011) Thymoquinone

Acetaminophen induced hepatotoxicity
Reduced in ALT activity

Anti-bacterial and anti-schistosomiasis effects
The effect of N. sativa seed (0%, 1%, 2% and 3% of diet) on performance, intestinal Escherichia coli (E. coli) colonization and jejunal morphology in laying hens was evaluated. The results showed that ileal E. coli numeration reduced with 1% N. sativa. However, the best intestinal health indices were obtained following administration of 2% N. sativa (Boka et al., 2014).
The effect of TQ (10 mg/kg, i.p.) against bacterial translocation and inflammatory responses induced by mechanical intestinal obstruction was studied in rats. The results indicated that TQ decreased inflammatory cytokines, oxidative damage, bacterial translocation and improved intestinal barrier function in rats with intestinal obstruction (Kapan et al., 2012).
In a clinical trial, the effect of N. sativa seed in comparison with a triple therapy including clarithromycin, amoxicillin, and omeprazole against Helicobacter pylori (H. pylori) was evaluated in patients with nonulcer dyspepsia. Patients were randomly divided into four groups: I) Triple therapy; II) 1 g/day N. sativa + 40 mg omeprazole; III) 2 g/day N. sativa + 40 mg omeprazole and IV) 3 g/day N. sativa + 40 mg omeprazole for four weeks. The results indicated that 2 g/day N. sativa + 40 mg omeprazole has the best therapeutic effect on H. pylori activity (Salem et al., 2010).
The antioxidant and anti-schistosomal effects of garlic aqueous extract (125 mg kg -1, i.p.) and N. sativa oil (0.2 mg kg, i.p.) in normal mice and Schistosoma mansoni (S. mansoni)-infected mice were studied. Hematological parameters and levels of MDA, GSH, LDH, AST, and ALT were assessed in the liver. The results revealed that garlic extract and N. sativa oil reversed most of the hematological and biochemical changes and markedly improved the antioxidant capacity of treated infected mice as compared to untreated infected mice (Shenawy et al., 2008).
The effect of oral administration of N. Sativa oil (2.5 and 5 ml/kg) alone or in combination with praziquantel on liver injury induced by S. mansoni was investigated in mice. The results showed that N. Sativa oil reduced the number of S. mansoni worms in the liver and decreased the total number of ova deposited in both the liver and intestine. When N. Sativa oil was administered in combination with praziquantel, the most prominent effect was a further reduction in the dead ova number more than that induced by praziquantel alone (Mahmoud et al., 2002).
The schistosomicidal effect of N. sativa seed against S. mansoni miracidia, cercariae, and adult worms was evaluated. Findings showed that N. sativa seed had a strong biocidal effect against all stages of the parasite life and showed inhibitory effect on egg-laying in adult female worms. The results also indicated that N. sativa seed induced oxidative stress against adult worms which was determined by reduction in the activities of antioxidant enzymes (Mohamed et al., 2005).
The anti-bacterial and antischistosomiasis effects of N. sativa and TQ were summarized in Table 3. Table 3. Anti-bacterial and anti-schistosomiasis effect of N. sativa and thymoquinone in GI tract

N. sativa seed Intestinal escherichia coli colonization
Reduced numeration of ileal E. coli (Boka et al., 2014) N. sativa seed Schistosomamansoni infection Biocidal effect in all stages of the parasite Inhibitory effect on egg-laying of adult female worms (Mohamed et al., 2005) N. sativa seed Patients with H. pylori Potential effect on H. pylori activity (Salem et al., 2010)

N. sativa oil
Schistosomamansoni infection Reduced the number of S. mansoni worms in the liver Reduced the total number of ova (Mahmoud et al., 2002) N. sativa oil Schistosomamansoni infection Inhibited most of the hematological and biochemical changes Improved the antioxidant capacity (Shenawy et al., 2008)

Anti-inflammatory and antioxidant effects
The effects of N. sativa oil (0.88 g/kg, orally) on gastric secretion and ethanolinduced ulcer in adult male rats were assayed. The results showed that N. sativa oil increased gastric mucin content, free acidity and glutathione level, and decreased gastric mucosal histamine content. It is concluded that N. sativa oil has a protective effect on ethanol-induced ulcer (El-Dakhakhny et al., 2000).
In another study, gastroprotective effects of N. sativa oil (2.5 and 5 ml/kg, orally) and TQ (5, 20, 50 and 100 mg/kg, orally) against gastric mucosal injury induced by ischaemia/reperfusion were evaluated in male Wistar rats. The results indicated that N. sativa oil and TQ at 5 and 20 mg/kg reduced LDH, LPO and increased GSH and SOD. It is concluded that N. sativa oil and TQ had a protective effect on gastric injury (El-Abhar et al., 2003).
The effects of N. sativa oil (10 mL/kg body weight, orally) and TQ (10 mg/kg body weight, orally) against acute alcoholinduced gastric mucosal injury were investigated in male albino rats. The findings showed that N. sativa oil caused a reduction in ulcer index and MDA level and promoted healing of gastric injury and SOD, GSH and GST levels. Likewise, TQ has a protective activity on gastric lesions but less than that of N. sativa (Kanter et al., 2005b) The gastroprotective and anti-secretory effects of N. sativa seed powder (1.0, 1.5 and 2.0 g/kg, oral), aqueous and ethanolic extracts of N. sativa seed powder (2.0 g/kg, oral), and N. sativa ethanol-ethyl acetate fraction (2.0 g/kg, oral) were investigated in indomethacin-treated rats. The results showed that N. sativa seed powder decreased indomethacin-induced gastric lesions in a dose-dependent manner. Ethanolic extract of N. sativa significantly reduced gastric secretion volume, pH, acidoutput and ulcer index, whereas aqueous extract only decreased gastric acid-output (Rifat-uz-Zaman and Khan, 2004).
In another study, the protective effect of N. sativa oil (10 ml/kg body weight) against piroxicam-induced gastric mucosal injury in adult male albino rats was investigated using light and scanning electron microscope. The results showed that N .sativa oil improved the structure of the mucosa in rats that received piroxicam and increased mucus secretion (Mohammed et al., 2010). The protective effect of N. sativa oil (10 ml/kg body weight) on stress-induced gastric ulcer in hypothyroid rats was studied. Animals were randomly divided into six groups: I) Control; II) Surgically thyroidectomized group; III) Acute cold restraint stressed group; IV) Surgically thyroidectomized and stressed group; V) N. sativa oil group and VI) Surgically thyroidectomized and stressed receiving N. sativa oil group. Findings indicated a reduction in thyroid hormone level and an increase in stress-induced gastritis which can be inhibited by N. sativa oil (Abdel Sater, 2009).
The effects of two-week administration of N. Sativa oil (0.88 mL/kg/day, orally), omeprazole (30 mg/kg body weight/day, orally) and corn oil (2 mL/kg/day, orally) on ethanol-induced gastric lesions were studied in rats. The results indicated that N. sativa oil significantly increased glutathione and antioxidant enzymes and decreased lipid peroxides and protein carbonyl content. It is concluded that coadministration of omeprazole and N. sativa oil significantly improved all of the studied parameters (El-Masry et al., 2010).
In one study, the effects of TQ (10 and 20mg/kg), omeprazole (10 and 20mg/kg) or co-administration of TQ (10mg/kg) and omeprazole (10mg/kg) on gastric mucosal ischemia/reperfusion injury induced by pyloric ligation (30 min), ischemia (30 min)/reperfusion (120 min) were investigated in rats. The results revealed that TQ had gastroprotective effects which were mediated by inhibiting proton pump, acid secretion and neutrophil infiltration, and increasing mucin secretion, and nitric oxide production (Magdy et al., 2012).
The antioxidative and antihistaminergic effects of N. sativa (500mg/kg, oral) and TQ (10mg/kg, orally) on ethanol-induced gastric mucosal damage were investigated in rat. The results showed that N. sativa significantly decreased the number of mast cells, the area of gastric erosions, histamine levels and myeloperoxidase activity. However, TQ effect was less pronounced as compared to that of N. sativa. The results also suggested that gastroprotective effects of N. sativa could be due to its anti-peroxidative, antioxidant and anti-histaminergic effects (Kanter et al., 2006).
The effect of N. seed oil (2.5 ml/kg, orally) on gastric tissues in experimental colitis (trinitrobenzenesulphonic acidinduced colitis) was studied. The levels of sialic acid (SA), GSH, MDA and CAT and SOD activities in gastric tissue samples and TNF-α, IL-1β and IL-6 and LDH levels in blood samples were determined. N. sativa seed oil significantly increased gastric tissue CAT activity and decreased LDH activity and TNF-α, IL-1β, IL-6 levels. Findings of this study indicated that N. sativa seed oil has a modulatory effect on inflammatory response in colitis (Emekli-Alturfan et al., 2011).
The effect of TQ (5 and 10 mg/kg) and sulfasalazine (500 mg/kg) as an anticolitis drug on acetic acid-induced colitis (by intracolonic injection of 3% acetic acid) was investigated in rats. Findings revealed that TQ has a more pronounced protective effect on colitis as compared to sulfasalazine and this effect may be possibly mediated through its antioxidant action (Mahgoub, 2003).
The effect of TQ (100 mg/kg, orally) on chronic pancretitis induced by high fat diet and ethanol was studied in rats. Findings revealed that TQ has a protective effect on pancretitis via reducing the secretion of amylase and lipase from pancreas, inflammatory cytokine and lipid peroxidation (Suguna et al., 2013).

Thymoquinone
Gastric mucosal ischemia/reperfusion (I/R) injury Inhibited proton pump, acid secretion and neutrophil infiltration Increased mucin secretion, and nitric oxide production (Magdy et al., 2012) Thymoquinone Chronic pancretitis induced by high fat diet and ethanol Reduced the secretion of amylase and lipase from pancreas, inflammatory cytokine and lipid peroxidation (Suguna et al., 2013) Conclusion N. sativa and its main constituent, TQ showed anti-cancer, hepatoprotective, antibacterial, anti-schistosomiasis, gastroprotective, anti-inflammatory and antioxidant effects in animal models of gastrointestinal disorders including cancers, hepatotoxicity, ischemia/reperfusion injury, cholestatic liver, non-ulcer dyspepsia, schistosomiasis infection, colitis and panceratitis. These effects supported the preventive and therapeutic effect of N. sativa and its constituents on inflammatory, oxidative and toxic injury due to various toxins, microbes and food allergens. Clinical studies also indicated preventive effect as well as relieving effect of this plant and its constituents on various gastrointestinal disorders. Therefore, N. sativa have both preventive and therapeutic effects on gastro-intestinal diseases.
However, further clinical and experimental investigations are required to reveal the exact perspective of molecular and cellular basis of the effects of N. sativa and its constituents.